EP0804589A1 - Tyrosine-kinase receptrice neuronale - Google Patents

Tyrosine-kinase receptrice neuronale

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Publication number
EP0804589A1
EP0804589A1 EP95917841A EP95917841A EP0804589A1 EP 0804589 A1 EP0804589 A1 EP 0804589A1 EP 95917841 A EP95917841 A EP 95917841A EP 95917841 A EP95917841 A EP 95917841A EP 0804589 A1 EP0804589 A1 EP 0804589A1
Authority
EP
European Patent Office
Prior art keywords
protein
nuk
tyrosine kinase
receptor tyrosine
substance
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP95917841A
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German (de)
English (en)
Inventor
Anthony Pawson
Mark Apartment 503 HENKEMEYER
Kenneth Letwin
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Mount Sinai Hospital Corp
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Mount Sinai Hospital Corp
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Publication date
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Publication of EP0804589A1 publication Critical patent/EP0804589A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to a novel receptor tyrosine kinase protein and isoforms and parts thereof, nucleic acid molecules encoding the novel protein and fragments thereof, and uses of the protein and nucleic acid molecules.
  • Embryonic development of multicellular organisms is a highly ordered process that requires coordination of individual cells. Every cell must decipher the numerous signals it receives and then properly execute commands in order to achieve the correct position and differentiated state in the animal. The extraordinar controls over cell growth, determination, migration and adhesion are mediated by molecules located on the plasma membrane surface.
  • a class of membrane associated molecules known to regulate cellular interactions are receptor tyrosine kinase proteins.
  • the evolutionary conservation of genes encoding receptor tyrosine kinase proteins and their targets has emphasized the importance of these proteins in intracellular communication, and has also provided model systems for genetic analysis of tyrosine kinase signalling pathways.
  • Such studies have shown that some tyrosine kinases function to specify a particular cell fate, such as the sevenless (sev) receptor in Drosophila R7 photoreceptor cells and the Let-23 receptor in nematode vulval cells (reviewed by Greenwald and Rubin, Cell 68:271-281, 1992).
  • Eph/Elk Eck subfamily is made up of at least fifteen related but unique gene sequences in higher vertebrates (Hirai et al, Science 238:1717-1720, 1987; Letwin et al, Oncogene 3:621-627, 1988; Lindberg et al., Mol. Cell Biol 10:6316-6324, 1990; Lhotak et al, Mol. Cell.
  • Eph family members encode a structurally related cysteine rich extracelluar domain containing a single immunoglobulin (Ig)-like loop near the N-terminus and two nbronectin L (FN El) repeats adjacent to the plasma membrane.
  • Eph family members include Cek5 (Pasquale, Cell Regulation 2:523-534, 1991) and Erk; (Chan and Watt, Oncogene 6:1057-1061 1991).
  • Another Eph family member, Sek has been shown to be segmentally expressed in specific rhombomeres of the mouse hindbrain (Nieto et al, Development 116:1137-1150, 1992). The presence of cell adhesion-like domains in this family of tyrosine kinases suggests that these proteins function in cell-cell interactions.
  • the other major families of proteins implicated in cell adhesion include the cadherins, selectins, integrins, and those of the immunoglobulin superfamily (reviewed by Hynes, R.O. and Landers, A.D., Cell 68, 303-322, 1992).
  • the extracelluar regions of cell adhesion molecules frequently contain peptide repeats, such as FN HI motifs, epidermal growth factor (EGF) repeats, or Ig loops that may direct protein-protein interactions at the cell surface.
  • FN HI motifs such as FN HI motifs, epidermal growth factor (EGF) repeats, or Ig loops that may direct protein-protein interactions at the cell surface.
  • EGF epidermal growth factor
  • a critical stage in the development of the nervous system is the projection of axons to their targets. Navigational decisions are made at the growth cones of the migrating axons. As axons grow their growth cones extend and retract filopodia and lamellipodia processes which are implicated in the navigational decisions and pathfinding abilities of migrating axons. Like peripheral nervous system axons, the growth cones of neurons associated with the central nervous system follow stereotyped pathways and apparently can selectively chose from a number of possible routes (reviewed by Goodman and Shatz, Cell 72:77-98, 1993). Early pathways in the vertebrate embryonic brain are thought to be arranged as a set of longitudinal tracts connected by commissures. However, the molecular mechanisms that underly growth cone navigation and axon pathfinding in development are poorly understood (Hynes, R.O. and Lander, A.D., 1992, Cell 68:303).
  • the present inventors have identified and characterized a receptor tyrosine kinase protein that plays an important role in cell-cell interactions and axonogenesis in the development of the nervous system.
  • the present inventors have cloned a novel murine gene, designated neural kinase (Nuk).
  • the gene encodes a new member of the Eph subfamily of receptor tyrosine kinases, designated Nuk protein.
  • the murine Nuk locus was mapped to the distal end of mouse chromosome 4 near the ahd-1 mutation.
  • Nuk protein The biological function of Nuk protein was investigated using antibodies having anti-Nuk protein specificity.
  • Nuk protein was also found to be concentrated at sites of cell-cell contact, often involving migrating neuronal cells or their extensions. Most notably, high levels of Nuk protein were found within initial axon outgrowths and associated nerve fibers, including most if not all peripheral nervous system (PNS) axons. The axonal localization of Nuk protein was also found to be transient and was not detected after migrations have ceased.
  • PNS peripheral nervous system
  • the present invention therefore provides a purified and isolated nucleic acid molecule containing a sequence encoding a receptor tyrosine kinase protein which is expressed in migrating axons, or an oligonucleotide fragment of the sequence which is unique to the receptor tyrosine kinase protein.
  • the purified and isolated nucleic acid molecule comprises (a) a nucleic acid sequence encoding a protein having the amino acid sequence as shown in SEQ ID NO:2 and Figure 2; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences which are at least 97% identical to (a); or, (d) a fragment of (a) or (b) that is at least 15 bases and which will hybridize to (a) or (b) under stringent hybridization conditions.
  • the purified and isolated nucleic acid molecule comprises (a) a nucleic acid sequence as shown in SEQ ID NO:l and Figure 1, wherein T can also be U; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences which are at least 85% identical to (a); or, (d) a fragment of (a) or (b) that is at least 15 bases and which will hybridize to (a) or (b) under stringent hybridization conditions.
  • a nucleic acid molecule of the invention may be prepared having deletion and insertion mutations.
  • the extracelluar domain or parts thereof such as the FN HI and Ig domains; the transmembrane region or parts thereof; the tyrosine kinase domain or parts thereof, such as the ATP binding site and; the carboxy terminal tail may be deleted.
  • the deletions are in a portion of the nucleic acid molecule of the invention encoding the extracelluar domain of Nuk protein, most preferably the portion comprising codons 29 to 50 in SEQ ID NO:l.
  • the deletions are in a portion of the nucleic acid sequence of the invention encoding the kinase domain of Nuk protein, most preferably the portion comprising the ATP-binding site amino acid number 623-707.
  • the invention further contemplates a purified and isolated double stranded nucleic acid molecule containing a nucleic acid molecule of the invention or a fragment thereof, hydrogen bonded to a complementary nucleic acid base sequence.
  • nucleic acid molecules of the invention may be inserted into an appropriate expression vector, i.e. a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
  • an appropriate expression vector i.e. a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
  • recombinant DNA molecules adapted for transformation of a host cell may be constructed which comprise a nucleic acid molecule of the invention and one or more transcription and translation elements operatively linked to the nucleic acid molecule.
  • a recombinant molecule which contains a nucleic acid molecule of the invention having a deletion or insertion mutation.
  • a recombinant molecule may further comprise a reporter gene.
  • the recombinant molecule can be used to prepare transformed host cells expressing the protein or part thereof encoded by a nucleic acid molecule of the invention. Therefore, the invention further provides host cells containing a recombinant molecule of the invention.
  • the invention also contemplates transgenic non-human mammals whose germ cells and somatic cells contain a recombinant molecule of the invention.
  • the invention further provides a method for preparing a novel receptor tyrosine kinase protein or isoforms or parts thereof utilizing the purified and isolated nucleic acid molecules of the invention.
  • the invention further broadly contemplates a purified and isolated receptor tyrosine kinase protein which is expressed in migrating axons, or an isoform or a part of the protein.
  • a purified receptor tyrosine kinase protein is provided which has the amino acid sequence as shown in SEQ ID NO:2 or Figure 2, or a sequence having between 97 and 100 percent identity thereto.
  • the receptor tyrosine kinase protein of the invention may also be phosphorylated.
  • Conjugates of Nuk protein of the invention, or parts thereof, with other molecules, such as proteins or polypeptides, may be prepared. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins.
  • a fusion protein is provided comprising a part of the protein of the invention, preferably the extracelluar domain, most preferably having the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 26 to 548 or amino acids 600 to 618; or the carboxy terminal, most preferably having the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 601 to 994; or sequences having at least 97% identity thereto.
  • the invention also permits the construction of nucleotide probes which are unique to the nucleic acid molecules of the invention and accordingly to the novel receptor tyrosine kinase protein of the invention or a part of the protein.
  • the invention also relates to a probe comprising a nucleotide sequence coding for a protein which displays the properties of the novel receptor tyrosine kinase of the invention or a part which is unique to the protein.
  • the probe may be labelled, for example, with a detectable substance and it may be used to select from a mixture of nucleotide sequences a nucleotide sequence coding for a protein which displays the properties of the novel receptor tyrosine kinase protein of the invention, or a part thereof.
  • the invention still further provides a method for identifying a substance which is capable of binding to the novel receptor tyrosine kinase protein of the invention, or an isoform or part of the protein, comprising reacting the novel receptor tyrosine kinase protein of the invention, or part of the protein, with at least one substance which potentially can bind with the receptor tyrosine kinase protein, isoform or part of the protein, under conditions which permit the formation of substance-receptor kinase protein complexes, and assaying for substance-receptor kinase protein complexes, for free substance, for non-complexed receptor kinase proteins, or for activation of the receptor tyrosine kinase proteins.
  • ligands are identified which are capable of binding to and activating the novel receptor tyrosine kinase protein of the invention.
  • the extracellular ligands which bind to and activate the novel receptor tyrosine kinase protein of the invention may be identified by assaying for protein tyrosine kinase activity.
  • the invention provides a method for assaying a medium for the presence of an agonist or antagonist of the interaction of a receptor tyrosine kinase protein of the invention and a substance which binds to the receptor tyrosine kinase protein, preferably a ligand.
  • the method comprises providing a known concentration of a receptor tyrosine kinase protein of the invention, isoforms thereof, or part of the protein, preferably the extracelluar domain of the protein, incubating the receptor tyrosine kinase protein with a substance which is capable of binding to the receptor tyrosine kinase protein, isoforms thereof, or part of the protein, and a suspected agonist or antagonist substance under conditions which permit the formation of substance-receptor protein complexes, and assaying for substance-receptor protein complexes, for free substance, for non-complexed proteins, or for activation of the receptor tyrosine kinase protein.
  • the methods of the invention permit the identification of potential stimulators or inhibitors of axonal migration and nerve cell interactions in development and regeneration, which will be useful in the treatment of nerve disorders and nerve damage.
  • the invention also contemplates a method for identifying a substance which is capable of binding to an activated receptor tyrosine kinase protein of the invention, or an isoform or part of the activated protein, comprising reacting the activated receptor tyrosine kinase protein of the invention, or part of the protein, with at least one substance which potentially can bind with the receptor tyrosine kinase protein, isoform or part of the protein, under conditions which permit the formation of substance-receptor kinase protein complexes, and assaying for substance-receptor kinase protein complexes, for free substance, for non-complexed receptor kinase proteins, or for phosphorylation of the substance.
  • intracellular ligands such as Src homology region 2 (SH2)-containing proteins which are capable of binding to a phosphorylated receptor tyrosine kinase protein of the invention, or intracellullar ligands which may be phosphorylated by the novel receptor tyrosine kinase of the invention may be identified.
  • the invention further contemplates antibodies having specificity against an epitope of the receptor tyrosine kinase protein of the invention or part of the protein which is unique to the receptor tyrosine kinase protein.
  • Antibodies may be labelled with a detectable substance and they may be used to detect the novel receptor tyrosine kinase of the invention in tissues and cells. The antibodies may accordingly be used to monitor axonal migration and nerve cell interactions.
  • Substances which affect axonal migration may be identified using the methods of the invention by comparing the pattern and level of expression of the novel receptor tyrosine kinase protein of the invention in tissues and cells in the presence and in the absence of the substance.
  • the invention provides a method for screening for substances having pharmaceutical utility in the treatment and diagnosis of nerve disorders and nerve damage.
  • Figure 1 shows the nucleotide sequence encoding the novel receptor tyrosine kinase protein of the invention as shown in SEQ ID NO: 1;
  • Figure 2 shows the amino acid sequence of the novel receptor tyrosine kinase protein of the invention as shown in SEQ ID NO:2 and a schematic diagram of the regions of the novel receptor tyrosine kinase protein of the invention;
  • Figure 3A shows immunoprecipitation of the novel receptor tyrosine kinase protein of the invention
  • Figure 3B shows Western Blot analysis of immunoprecipitates of the novel receptor tyrosine kinase protein of the invention
  • Figure 4A shows localization of mRNA of the novel receptor tyrosine kinase protein of the invention in whole-mount sections of 7.5 day old mouse embryo;
  • Figure 4B shows localization of the novel receptor tyrosine kinase protein of the invention in whole-mount sections of 8 day old mouse embryo;
  • Figure 4C shows localization of the novel receptor tyrosine kinase protein of the invention in whole-mount sections of 8.75 day old mouse embryo;
  • Figure 4D shows localization of the novel receptor tyrosine kinase protein of the invention in whole-mount sections of 9.5 day old mouse embryo;
  • Figure 4E shows localization of the novel receptor tyrosine kinase protein of the invention in whole-mount sections of 9.5 day old mouse embryo in greater detail than in Figure 4D;
  • Figure 4F shows localization of the novel receptor tyrosine kinase protein of the invention in paraffin serial transverse sections of 9.5 day old mouse embryo;
  • Figure 4G shows localization of the novel receptor tyrosine kinase protein of the invention in paraffin serial transverse sections of 9.5 day old mouse embryo;
  • Figure 4H shows localization of the novel receptor tyrosine kinase protein of the invention in paraffin sagittal section along the midline of 10.5 day old mouse embryo;
  • Figure 41 shows localization of the novel receptor tyrosine kinase protein of the invention in paraffin adjacent transverse sections of 10.5 day old mouse embryo immunoreacted with anti-Nuk protein antibodies
  • Figure 4J shows localization of the novel receptor tyrosine kinase protein of the invention in paraffin adjacent transverse sections of 10.5 day old mouse embryo immunoreacted with a trpE-Nuk peptide;
  • Figure 5A shows an adjacent transverse section of an 11.5 day embryo at the level of the caudal/posterior spinal cord immunoreacted with anti-Nuk antibodies
  • Figures 5B shows an adjacent transverse section of an 11 day embryo at the level of the caudal/posterior spinal cord immunoreacted with anti-Nuk antibodies preincubated with a trpE-Nuk peptide;
  • Figures 5C shows transmission electron microscopy immunolocalization of Nuk protein in ventral midbrain cells of a 9.5 day embryo
  • Figure 5D shows transmission electron microscopy (EM) immunolocalization of Nuk protein in ventral midbrain cells of a 9.5 day embryo
  • Figure 6A is a photomicrograph of the head of an 11.5 day anti-Nuk whole-mount immunostained embryo with one of the pair of oculomotor nerve fibers in focus showing strong labelling for Nuk protein (filled arrow);
  • Figure 6B shows a clearer view of Nuk protein staining in the oculomotor axons fibers obtained when the whole-mount staged embryo was filleted down the midline to minimize tissue thickness;
  • Figure 6C shows a paraffin section of a 10.5 to 11 day mouse embryo immunostained with anti-Nuk
  • Figure 6D shows a paraffin section of a 10.5 to 11 day mouse embryo immunostained with anti-Neurofilament antibodies.
  • Figure 6E is a frontal section immunostained with anti-Nuk antibodies which label the ventral midbrain and oculomotor axon fibers as they exit the neural tube (filled arrow) and extend (open arrow) towards their target tissue, the pre-optic muscle mass;
  • Figure 6F shows a sagittal section immunostained with anti-Nuk antibodies which label the oculomoter axon fascicule as it enters the pre-optic muscle mass (open arrow);
  • Figure 7A is a 10.5 day whole-mount embryo immunostained with anti-Nuk antibodies which label the trigeminal nerve V and facial nerve VII;
  • Figures 7B shows transverse sections of a 10.5 day embryo showing Nuk-positive trigeminal nerve V axon fascicules labelled with the anti-Nuk antibodies;
  • Figures 7C shows transverse sections of a 10.5 day embryo showing
  • Figures 7D is an 11.5 day anti-Nuk whole-mount showing localization of Nuk protein in the vagus nerve X associated fibers as they pathfind to their target visceral organs (curved open arrows);
  • Figures 7E is an 11.5 day anti-Nuk whole-mount showing localization of Nuk protein in the vagus nerve X associated fibers as they pathfind to their target visceral organs (curved open arrows);
  • Figure 7F is a whole-mount 10 day embryo showing Nuk protein concentrated within the earliest spinal nerve fibers exiting the neural tube (arrows);
  • Figure 7G shows a slightly later stage embryo from that shown in Figure 7F;
  • Figures 7H shows a transverse section bisecting the rostral spinal cord of an 11 day embryo demonstrating that the darkly stained Nuk-positive fibers shown in Figures 7F and 7G are ventral motor axons (open arrows).
  • Figures 71 shows a transverse section bisecting the rostral spinal cord of an 11 day embryo demonstrating that the darkly stained Nuk-positive fibers shown in Figures 7F and 7G are ventral motor axons (open arrows).
  • Figures 7J shows both sides of an 11 day whole-mount embryo demonstrating that Nuk protein is localized within the spinal motor nerves as they elongate to the plexus regions (open arrows);
  • Figures 7K shows both sides of an 11 day whole-mount embryo demonstrating that Nuk protein is localized within the spinal motor nerves as they elongate to the plexus regions;
  • Figure 7L shows a close-up of a 12 day whole-mount embryo immunostained with anti-Nuk antibodies which label the nerve fibers of the parasympathetic chain ganglion;
  • Figure 8A shows a whole-mount 10.5 day embryo showing Nuk protein localization (arrow) at the dorsal region of the otic vesicle (ov) surrounding the budding endolymphatic duct;
  • Figure 8B shows a whole-mount 11.5 day embryo showing elongation of the endolymphatic duct;
  • Figure 8C shows a whole-mount 11.5 day embryo at a slightly different focal plane from Figure 8B showing that the vestibulocochlear sensory fibers connecting to the developing ear stain positive for Nuk protein (arrow);
  • Figure 8D shows a transverse section of an 11.5 day embryo showing high levels of Nuk protein localized to the basement membrane of the endolymphatic duct cells where they contact the surrounding mesenchymal cells (open arrow;
  • Figure 9 shows a recombinant DNA molecule of the invention having a null mutation obtained by deletion of exon 2, corresponding to codons 29 to 50 as shown in SEQ ID NO: 1;
  • Figure 10 shows a recombinant DNA molecule of the invention encoding the N u fi mutation in the ATP binding region of the kinase domain of Nuk protein, and a lac Z reporter gene;
  • Figure 11A shows expression of the Nuk 2 mutation in a mouse embryo at the six somite stage (8.25 days development) in the brain and developing heart;
  • Figure 11B shows expression of the N k 2 mutation in a mouse embryo at the 14 somite stage (8.75 days development) in the hindbrain rhombomeres, the midbrain, diencephalon and in the heart
  • Figure 12A shows Nuk 2 expression in a 10.5 day old mouse embryo in the ventral midbrain, dienchephalon and retinal cells;
  • Figure 12B shows Nuk 2 expression in a 10.5 day old mouse embryo in the brain and spinal cord
  • Figure 13 shows an immunoblot illustrating that Nuk protein autophosphorylation is induced by Elk-ligand stimulation
  • Figure 14 is an immunoblot showing expression of Nuk protein in cell lines
  • Figure 15 is an immunoblot showing binding of phosphorylated/non-phosphorylated Nuk protein to SH2-containing GST- fusion proteins
  • Figure 16 is an immunoblot showing binding of phosphorylated/non-phosphorylated Nuk protein to SH2-containing GST- fusion proteins
  • Figure 17 is an immunoblot showing binding of Nuk Protein to SH2- containing GST-fusion proteins in the presence of a competing phosphorylated peptide
  • Figure 18 shows immunoblots illustrating the phosphorylation of proteins after ELK-ligand stimulation of COS cells.
  • the present inventors have identified and sequenced a nucleic acid molecule encoding a novel receptor tyrosine
  • ISA/EP kinase protein with a unique expression pattern as described herein.
  • the receptor tyrosine kinase protein of the invention is also referred to herein as Neural Kinase (Nuk) protein.
  • the Nuk coding region was cloned using a ⁇ gtlO expression library constructed from mouse embryo mRNA. The library was probed with a partial n Ql Nuk cDNA insert. Additional 5'Nuk coding sequences were obtained by rapid amplification of cDNA ends (RACE). Translation of combined RACE and cDNA clones revealed a single open reading frame of 994 codons.
  • the Nuk locus mapped to the distal end of mouse chromosome 4 near the QM mutation.
  • the Nuk protein belongs to the EphlElklEck family, of which many members are expressed in the developing nervous system.
  • the protein encoded by the deduced amino acid sequence of Nuk has all the hallmarks of an Eph family member, including a number of conserved residues of the Eph family, for example the 20 cysteine residues whose position is conserved in the extracelluar domain of Eph family members (bold type, Figure 2), an immunoglobulin-like domain near the amino terminus (Ig-like), and two fibronectin type HI repeats (FN HI); between Nuk residues 330-420 and 444-534.
  • the Ig-like domain of Nuk protein contains specific residues (Cys 70 , Trp 80 , Cys 115 ) known to be conserved in the Ig superfamily (Williams and Barclay, Ann. Rev. Immunol 6:381-405, 1988). When compared to other known members of the Eph family, Nuk protein was found to be most highly related to the full length amino acid sequence of chicken Cek5 (96% identity).
  • a purified and isolated nucleic acid molecule containing a sequence encoding a protein having the amino acid sequence as shown in SEQ ID NO:2 and Figure 2.
  • the purified and isolated nucleic acid molecule of the invention contains a nucleic acid sequence as shown in SEQ ID NO:l and Figure 1.
  • Fragments of the nucleic acid molecules are contemplated by the present invention.
  • the fragments include fragments of the nucleotide sequence as shown in SEQ. ID. NO. 1 and in Figure 1 that have at least 15 bases to 18 bases, preferably at least 15 bases, and which are capable of hybridizing to the nucleotide sequence as shown in SEQ ID NO.
  • a double stranded nucleotide sequence comprising a nucleic acid molecule of the invention or a fragment thereof, hydrogen bonded to a complementary nucleotide base sequence, and an RNA made by transcription of this double stranded nucleotide sequence are contemplated by the present invention.
  • sequences having substantial sequence identity means those nucleic acid and amino acid sequences which have slight or inconsequential sequence variations from the sequences disclosed in Figures 1 and 2 and SEQ ID NOS: 1 and 2, i.e. the homologous sequences function in substantially the same manner to produce substantially the same polypeptides as the actual sequences.
  • the variations may be attributable to local mutations or structural modifications.
  • Nucleic acid sequences having substantial identity include nucleic acid sequences which encode proteins having at least 97% sequence identity with the amino acid sequences as shown in SEQ. ID. NO:2 and in Figure 2; nucleic acid sequences having at least 85%, preferably at least 90%, most preferably at least 95% identity with the nucleic acid sequence as shown in SEQ. ID. NO.:l and in Figure 1; and fragments thereof having at least 15 to 18, preferably at least 15 bases which will hybridize to these sequences under stringent hybridization conditions.
  • Stringent hybridization conditions are those which are stringent enough to provide specificity, reduce the number of mismatches and yet are sufficiently flexible to allow formation of stable hybrids at an acceptable rate.
  • the invention further provides amino acid sequences which have substantial identity with the amino acid sequence shown in SEQ ID NO:2 and in Figure 2. Substantially identical sequences include sequences having at least 97% sequence identity.
  • the invention still further provides peptides which are unique to the receptor tyrosine kinase protein of the invention. Preferably, the peptides have at least 10 to 20 amino acids.
  • the sequence of the nucleic acid molecule of the invention or a fragment thereof, may be inverted relative to its normal presentation for transcription to produce antisense nucleic acid molecules.
  • the antisense nucleic acid molecules may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • the antisense nucleic acid molecules may be used in gene therapy to treat inherited disorders of the nervous system.
  • a number of unique restriction sequences for restriction enzymes are incorporated in the nucleic acid sequence identified in SEQ ID NO: 1 and in Figure 1 and these provide access to nucleic acid sequences which code for polypeptides unique to the receptor tyrosine kinase protein of the invention.
  • Nucleic acid sequences unique to the receptor tyrosine kinase protein of the invention or isoforms or parts thereof, can also be constructed by chemical synthesis and enzymatic ligation reactions carried out by procedures known in the art.
  • the invention contemplates isoforms of the receptor tyrosine kinase protein of the invention.
  • An isoform contains the same number and kinds of amino acids as the protein of the invention, but the isoform has a different molecular structure.
  • the isoforms contemplated by the present invention are those having the same functional properties as the novel receptor tyrosine kinase protein of the invention as described herein.
  • the present invention also includes conjugates of the receptor tyrosine kinase protein of the invention, or parts thereof.
  • the receptor tyrosine kinase protein or portions thereof may be conjugated with a selected protein or marker protein to produce fusion proteins.
  • the present invention also includes a receptor tyrosine kinase protein of the invention or part thereof, preferably the catalytic domain, which is enzymatically active.
  • the catalytically active form of the protein or part thereof is also referred to herein as an "activated receptor tyrosine kinase protein or part thereof.
  • Nuk protein has been localized during early embryogenesis.
  • the restricted expression of Nuk imposes constraints on the cellular range of activity of the putative Nuk ligand, and indicates that the Nuk locus plays unique and important roles in the determination, migration and pathfinding of axons, in axogenesis and fasciculation, in neural tube formation, and in the regulation of specific cell-cell interactions during early development of the nervous system.
  • the Nuk locus also plays an important role in axonal migration during regeneration following injury to the peripheral nervous system.
  • Nuk protein is confined to the developing nervous system, where it marks segments along the axis of the neural tube in the hindbrain and specific morphological bulges of the midbrain and forebrain. Nuk is expressed in a rhombomere-specific pattern early during hindbrain segmentation.
  • the restriction of Nuk protein to specific anterior-posterior and dorsal- ventral compartments during early development of the rostral neural tube indicates this receptor tyrosine kinase protein functions in the patterning of specific brain structures.
  • Nuk protein was found to be expressed in the developing nervous system and, in particular, is highly expressed very early in the retinal ganglion cells and in the group of cells that form the optic chiasm just prior to axonogenesis of the retinal cells. These observations indicate that Nuk protein participates in early development of the visual system components and in pathfinding of retinal axons. The present inventors have also detected Nuk in cells of the ventral midbrain and in the endolymphatic duct of the developing ear.
  • the present inventors have also localized Nuk protein to specific locations within the cells of the developing nervous system and have shown that Nuk protein is associated with the plasma membrane of migrating neural cells.
  • Nuk protein is concentrated at sites of cell-cell contact, of migrating neuronal cells or their extensions and high levels of Nuk protein are found within initial axon outgrowths and associated nerve fibers.
  • the axonal localization of Nuk is transient and is not detected after the growth cones have reached their targets and migrations have ceased, indicating a role for this receptor tyrosine kinase protein during the early migration, pathfinding and fasciculation stages of axonogenesis.
  • Nuk protein may function to transmit signals from the plasma membrane and may cooperate with other neuronal tyrosine kinases, such as Sek.
  • Nuk protein is localized to at least one CNS axon pathway.
  • the subcellular localization of Nuk protein is similar to that observed for vimentin and the extracelluar matrix molecule laminin (Liesi, EMBO 4:1163-1170, 1985) and it coincides with pathways of neuronal cell migration along the radial glial fibers (Hatten, Trends Neurosci. 13:179-184, 1990).
  • the specific subcellular localization of Nuk protein to the cell-cell contacts between the basement membrane of the endolymphatic duct cells and the surrounding mesenchyme /neural crest cells indicates Nuk protein functions to modulate this interaction.
  • the concentration of Nuk protein at sites of cell-cell contact indicates that its ligand is a membrane-associated molecule.
  • Nuk protein immunoreactivity was frequently observed on the membranes of both cells at the site of contact and was generally observed to localize to specific regions of the membrane (see Figures 5 and 8). Therefore, homophilic/heterophilic interactions between Eph receptors may play a role in their biological functions.
  • nucleic acid molecules of the invention encoding the novel receptor tyrosine kinase protein, or fragments thereof may be isolated and sequenced, for example, by synthesizing cDNAs from mouse embryo RNA and using rapid amplification of cDNA ends (RACE, Frohman, et al., Proc. Na+, Acad. Sd. USA, 85, 8998-9002, 1988) using oligonudeotides spedfic for the novel receptor tyrosine kinase protein, and analysing the sequences of the clones obtained following amplification.
  • RACE Rapid amplification of cDNA ends
  • Oligonu eotides specific for the novel receptor tyrosine kinase protein may be identified by comparing the nucleic acid sequence of the nudeic acid molecules of the invention to known sequences, for example, sequences of the other members of the Eph subfamily. Nucleic acid molecules of the present invention encoding the novel receptor tyrosine kinase protein and oligonucleotide fragments thereof, may also be constructed by chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • the novel tyrosine kinase receptor protein of the invention may be prepared using recombinant DNA methods. Accordingly, the nucleic add molecules of the present invention having a sequence which codes for the receptor tyrosine kinase protein of the invention, or a fragment thereof may be incorporated in a known manner into an appropriate expression vector which ensures good expression of the protein or part thereof. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses, so long as the vector is compatible with the host cell used.
  • the invention therefore contemplates a recombinant molecule of the invention containing a nucleic acid molecule of the invention, or a fragment thereof, and the necessary elements for the transcription and translation of the inserted protein-sequence.
  • Suitable transcription and translation elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Selection of appropriate transcription and translation elements is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art. Examples of such elements include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal.
  • transcriptional and translation elements may be incorporated into the expression vector.
  • additional DNA restriction sites such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring indudbility of transcription
  • sequences conferring indudbility of transcription may be incorporated into the expression vector.
  • the necessary transcriptional and translation elements may be supplied by the native receptor tyrosine kinase protein and/or its flanking regions.
  • the recombinant molecules of the invention may also contain a reporter gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention.
  • reporter genes are genes encoding a protein such as ⁇ -galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG.
  • the reporter gene is lac Z.
  • Transcription of the reporter gene is monitored by changes in the concentration of the reporter protein such as ⁇ -galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. This makes it possible to visualize and assay for expression of recombinant molecules of the invention and in particular to determine the effect of a mutation on expression and phenotype.
  • the reporter protein such as ⁇ -galactosidase, chloramphenicol acetyltransferase, or firefly luciferase.
  • Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation etc.
  • Methods for transforming transfecting, etc. host cells to express foreign DNA are well known in the art (see, e.g., Itakura et al., U.S. Patent No. 4,704,362; Hinnen et al., PNAS USA 75:1929-1933, 1978; Murray et al., U.S. Patent No. 4,801,542; Upshall et al., U.S. Patent No. 4,935,349; Hagen et al., U.S. Patent No. 4,784,950; Axel et al, U.S. Patent No.
  • Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells, including bacterial, mammalian, yeast or other fungi, viral, plant, or insect cells.
  • bacterial host cells suitable for carrying out the present invention indude E. coli, B. subtilis, Salmonella typhimurium, and various species within the genus' Pseudomonas, Streptomyces, and Staphylococcus, as well as many other bacterial species well known to one of ordinary skill in the art.
  • Representative examples of baderial host cells include DH5n (Stratagene, Lajolla, California), JM109 ATCC No. 53323, HB101 ATCC No. 33694, and MN294.
  • Suitable bacterial expression vectors preferably comprise a promoter which functions in the host cell, one or more selectable phenotypic markers, and a bacterial origin of replication.
  • Representative promoters include the ⁇ -ladamase (penicillinase) and lactose promoter system (see Chang et al., Nature 275:615, 1978), the trp promoter (Nichols and Yanofsky, Meth in Enzymology 101:155, 1983) and the tac promoter (Russell et al., Gene 20: 231, 1982).
  • Representative selectable markers include various antibiotic resistance markers such as the kanamycin or ampicillin resistance genes.
  • Suitable expression vectors include but are not limited to bacteriophages such as lambda derivatives or plasmids such as pBR322 (see Bolivar et al., Gene 2:9S, 1977), the pUC plasmids pUC18, pUC19, pUC118, pUC119 (see Messing, Meth in Enzymology 101:20-77, 1983 and Vieira and Messing, Gene 19:259-268, 1982), and pNH8A, pNH16a, pNH18a, and Bluescript M13 (Stratagene, La Jolla, Calif.).
  • Yeast and fungi host cells suitable for carrying out the present invention include, among others Saccharomyces cerevisae, the genera Pichia or Kluyveromyces and various species of the genus Aspergillus.
  • Suitable expression vectors for yeast and fungi include, among others, YC p 50 (ATCC No. 37419) for yeast, and the amdS cloning vector pV3 (Turnbull, Bio /Technology 7:169, 1989). Protocols for the transformation of yeast are also well known to those of ordinary skill in the art.
  • transformation may be readily accomplished either by preparation of spheroplasts of yeast with DNA (see Hinnen et al., PNAS USA 75:1929, 1978) or by treatment with alkaline salts such as LiCl (see Itoh et al., J. Bacteriology 153:163, 1983). Transformation of fungi may also be carried out using polyethylene glycol as described by Cullen et al. (Bio /Technology 5:369, 1987).
  • Mammalian cells suitable for carrying out the present invention include, among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573) and NS-1 cells.
  • Suitable expression vectors for directing expression in mammalian cells generally include a promoter, as well as other transcriptional and translational control sequences. Common promoters include SV40, MMTV, metallothionein-1, adenovirus Ela, CMV, immediate early, immunoglobulin heavy chain promoter and enhancer, and RSV-LTR.
  • Protocols for the transfection of mammalian cells are well known to those of ordinary skill in the art. Representative methods indude caldum phosphate mediated electroporation, retroviral, and protoplast fusion-mediated transfection (see Sambrook et al., supra).
  • promoters, terminators, and methods for introducing expression vectors of an appropriate type into plant, avian, and insect cells may also be readily accomplished.
  • Nuk or derivatives thereof may be expressed from plant cells (see Sinkar et al., J. Biosd (Bangalore) 11:47-58, 1987, which reviews the use of Agrobacterium rhizogenes vectors; see also Zambryski et al., Genetic Engineering, Principles and Methods, Hollaender and Setlow (eds.), Vol. VI, pp. 253-278, Plenum Press, New York, 1984, which describes the use of expression vectors for plant cells, including, among others, pAS2022, pAS2023, and pAS2034).
  • Insect cells suitable for carrying out the present invention include cells and cell lines from Bombyx or Spodotera species.
  • Suitable expression vectors for directing expression in insect cells include Baculoviruses such as the Autographa California nudear polyhedrosis, virus (Miller et al. 1987, in Genetic Engineering, Vol. 8 ed. Setler, J.K. et al., Plenum Press, New York) and the Bombyx mori nuclear polyhedrosis virus (Maeda et al., 1985, Nature 315:592).
  • Nuk may be expressed in non-human trans genie animals such as, mice, rats, rabbits, sheep and pigs (see Hammer et al. (Nature 315:680-683, 1985), Palmiter et al. (Sdence 222:809-814, 1983), Brinster et al. (Proc
  • Nuk protein or parts thereof may also be prepared by diemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and ⁇ , Thieme, Stuttgart).
  • Conjugates of the Nuk protein of the invention, or parts thereof, with other molecules, such as proteins or polypeptides may be prepared. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins.
  • fusion proteins may be prepared by fusing, through recombinant techniques, the N-terminal or C-terminal of Nuk protein or parts thereof, and the sequence of a selected protein or marker protein with a desired biological function.
  • the resultant fusion proteins contain Nuk protein or a portion thereof fused to the selected protein or marker protein as described herein.
  • proteins which may be used to prepare fusion proteins indude immunoglobulins and parts thereof sudi as the constant region of immunglobulin ⁇ l, and lymphokines such as gamma interferon, tumor necrosis factor, IL-1, IL-2,IL-3, H-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, GM-CSF, CSF-1 and G-CSF.
  • lymphokines such as gamma interferon, tumor necrosis factor, IL-1, IL-2,IL-3, H-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, GM-CSF, CSF-1 and G-CSF.
  • Sequences which encode the above-described proteins may generally be obtained from a variety of sources, including for example, depositories which contain plasmids encoding sequences including the American Type Culture Collection (ATCC, Rockville Maryland), and the British Biotechnology Limited (Cowley, Oxford England).
  • examples of such plasmids include BBG 12 (containing the GM-CSF gene coding for the mature protein of 127 amino acids), BBG 6 (which contains sequences encoding gamma interferon), ATCC No. 39656 (which contains sequences encoding TNF), ATCC No. 20663 (which contains sequences encoding alpha interferon,) ATCC Nos. 31902 and 39517 (which contains sequences encoding beta interferon), ATCC
  • Nuk is doned into an expression vector as a fusion gene with the constant region of human immunoglobulin ⁇ l.
  • the expression vectors pNUT ⁇ GH and pVL1393 are prepared for cloning by digestion with Smal followed by dephosphorylation by calf intestinal alkaline phosphatase. The linear produd is isolated after agarose gel electrophoresis.
  • the Nuk genes are then generated by polymerase chain reaction using the cloned Nuk cDNA as a template.
  • the Nuk fusion protein is synthesized from the extracelluar domain of Nuk protein, preferably amino adds 26 to 548, SEQ ID NO: 2 and Figure 2.
  • the Nuk fusion protein is synthesized from the carboxy terminal tail of Nuk protein, preferably amino acids 601 to 994, SEQ ID NO:2 and Figure 2.
  • the constant region of an immunoglobulin, such as human ⁇ l gene may be prepared, for example, from pUCB7Ig monomer. Briefly, the CH gene is isolated by digestion with Xbal which cuts at the 3' end of the gene followed by treatment with E. coli DNA polymerase I in the presence of all four dNTPs in order to create a blunt end. The plasmid is then digested with Bdl which cuts at the 5' end of the gene. The fragment containing the heavy chain gene is isolated after electrophoresis in an agarose gel. The fusion Nuk amplified fragment is inserted into each prepared vector along with the heavy chain fragment.
  • Orientation of the resulting plasmids is determined by PCR with one priming oligo which anneals to the vector sequence and the other priming oligo which anneals to the insert sequence.
  • appropriate restriction digests can be performed to verify the orientation.
  • the sequence of the fusion Nuk immunoglobulin constant region gene can be verified by DNA sequencing.
  • Phosphorylated receptor tyrosine kinase proteins of the invention may be prepared using the method described in Reedijk et al. The EMBO Journal 11(4):1365, 1992.
  • tyrosine phosphorylation may be induced by infecting bacteria harbouring a plasmid containing a nucleotide sequence of the invention or fragment thereof, with a ⁇ gtll bacteriophage encoding the cytoplasmic domain of the Elk tyrosine kinase.
  • Bacteria containing the plasmid and bacteriophage as a lysogen are isolated. Following induction of the lysogen, the expressed receptor protein becomes phosphorylated.
  • nucleotide probes for use in the detection of nucleotide sequences in biological materials.
  • a nucleotide probe may be labelled with a detectable substance such as a radioactive label which provides for an adequate signal and has suffident half-life sudi as 3 2P, 3H, 14C or the like.
  • detectable substances include antigens that are recognized by a specific labelled antibody, fluorescent compounds, enzymes, antibodies specific for a labelled antigen, and luminescent compounds.
  • An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nudeotide to be detected and the amount of nucleotide available for hybridization.
  • Labelled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.).
  • the nucleotide probes may be used to detect genes, preferably in human cells, that encode proteins related to or analogous to the receptor tyrosine kinase protein of the invention.
  • the nucleotide probes may therefore be useful in the diagnosis of disorders of the nervous system arising from mutations or alterations to the Nuk gene or a homologue thereof.
  • the receptor tyrosine kinase protein of the invention and portions thereof, for example amino acids of the carboxy terminal tail, preferably amino acids 601 to 994; or amino acids of the extracellular domain, preferably amino adds 26 to 548 (SEQ ID NO: 2 and Figure 2), may be used to prepare antibodies.
  • Antibodies having specifidty for Nuk protein may also be raised from fusion proteins created by expressing trpE-Nuk fusion proteins in baderia as described above.
  • antibodies are understood to indude monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab') 2 and recombinantly produced binding partners.
  • Antibodies are understood to be reactive against Nuk protein if they bind with a K a of greater than or equal to 10- 7 M. As will be appredated by one of ordinary skill in the art, antibodies may be developed which not only bind to Nuk protein, but which bind to a ligand of Nuk protein, and which also block the biological activity of Nuk protein. Such antibodies will be useful in the diagnosis and treatment of disorders of the nervous system and nerve damage. Polyclonal antibodies may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, various fowl, rabbits, mice, or rats.
  • Nuk protein is utilized to immunize the animal through intraperitoneal, intramuscular, intraocular, or subcutaneous injections, in conjunction with an adjuvant such as Freund's complete or incomplete adjuvant. Following several booster immunizations, samples of serum are collected and tested for reactivity to Nuk protein. Particularly preferred polyclonal antisera will give a signal on one of these assays that is at least three times greater than bad ground. Once the titer of the animal has reached a plateau in terms of its reactivity to Nuk protein, larger quantities of antisera may be readily obtained either by weekly bleedings, or by exsanguinating the animal.
  • an adjuvant such as Freund's complete or incomplete adjuvant.
  • Monoclonal antibodies may also be readily generated using conventional techniques (see U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993 which are incorporated herein by reference; see also Monodonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are also incorporated herein by reference).
  • a subject animal such as a rat or mouse is injected with Nuk protein.
  • the Nuk protein may be admixed with an adjuvant such as Freund's complete or incomplete adjuvant in order to increase the resultant immune response.
  • an adjuvant such as Freund's complete or incomplete adjuvant in order to increase the resultant immune response.
  • the animal may be reimmunized with another booster immunization, and tested for reactivity to Nuk protein using assays described above. Once the animal has plateaued in its reactivity to Nuk protein, it is sacrificed, and organs which contain large numbers of B cells such as the spleen and lymph nodes are harvested.
  • Cells which are obtained from the immunized animal may be immortalized by transfection with a virus such as the Epstein bar virus (EBV) (see Glasky and Reading, Hybridoma 8(4):377-389, 1989).
  • EBV Epstein bar virus
  • the harvested spleen and/or lymph node cell suspensions are fused with a suitable myeloma cell in order to create a "hybridoma" which secretes monoclonal antibody.
  • Suitable myeloma lines indude, for example, NS-1 (ATCC No. TIB 18), and P3X63 - Ag 8.653 (ATCC No. CRL 1580).
  • the cells may be placed into culture plates containing a suitable medium, such as RPMI 1640, or DMEM (Dulbecco's Modified Eagles Medium) (JRH Biosciences, Lenexa, Kansas), as well as additional ingredients, such as Fetal Bovine Serum (FBS, ie., from Hyclone, Logan, Utah, or JRH Biosdences).
  • a suitable medium such as RPMI 1640, or DMEM (Dulbecco's Modified Eagles Medium) (JRH Biosciences, Lenexa, Kansas)
  • FBS Fetal Bovine Serum
  • the medium should contain a reagent which selectively allows for the growth of fused spleen and myeloma cells such as HAT (hypoxanthine, aminopterin, and thymidine) (Sigma Chemical Co., St. Louis, Missouri).
  • the resulting fused cells or hybridomas may be screened in order to determine the presence of antibodies which are reactive against Nuk protein.
  • assays may be utilized to determine the presence of antibodies which are reactive against Nuk protein, including for example Countercurrent Immuno-Electrophoresis, Radioimmunoassays, Radioimmunoprecipitations, Enzyme-Linked Immuno-Sorbent Assays (ELISA), Dot Blot assays, Inhibition or Competition Assays, and sandwich assays (see U.S. Patent Nos. 4,376,110 and 4,186,530; see also Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). Following several clonal dilutions and reassays, a hybridoma produdng antibodies reactive against Nuk protein may be isolated.
  • mRNA is isolated from a B cell population, and utilized to create heavy and light chain immunoglobulin cDNA expression libraries in the ⁇ mmunoZap(H) and ⁇ mmunoZap(L) vectors. These vectors may be screened individually or co-expressed to form Fab fragments or antibodies (see Huse et al. supra; see also Sastry et al., supra). Positive plaques may subsequently be converted to a non-lytic plasmid which allows high level expression of monoclonal antibody fragments from I coli.
  • binding partners may also be constructed utilizing recombinant DNA techniques to incorporate the variable regions of a gene which encodes a specifically binding antibody.
  • the genes which encode the variable region from a hybridoma producing a monoclonal antibody of interest are amplified using nucleotide primers for the variable region. These primers may be synthesized by one of ordinary skill in the art, or may be purchased from commerdally available sources. Primers for mouse and human variable regions including, among others, primers for V ⁇ a VHb VHC/ VH ./ CHI/ V and CL regions are available from Stratacyte (La Jolla, Calif).
  • These primers may be utilized to amplify heavy or light chain variable regions, which may then be inserted into vectors such as ImmunoZAPTMH or ImmunoZAPTML (Stratacyte), respectively. These vectors may then be introduced into £. coli for expression. Utilizing these techniques, large amounts of a single-chain protein containing a fusion of the VH and VL domains may be produced (See Bird et al., Science 242:423-426, 1988). In addition, such techniques may be utilized to change a "murine" antibody to a "human” antibody, without altering the binding specifidty of the antibody.
  • suitable antibodies or binding partners may be isolated or purified by many techniques well known to those of ordinary skill in the art (see Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). Suitable techniques include peptide or protein affinity columns, HPLC or RP-HPLC, purification on protein A or protein G columns, or any combination of these techniques.
  • the polyclonal or monoclonal antibodies may be used to detect the receptor tyrosine kinase protein of the invention in various biological materials, for example they may be used in an ELISA, radioimmunoassay or histochemical tests. Thus, the antibodies may be used to quantify the amount of a receptor tyrosine kinase protein of the invention in a sample in order to determine its role in particular cellular events or pathological states and to diagnose and treat such pathological states.
  • polyclonal and monoclonal antibodies of the invention may be used in immuno-histochemical analyses, for example, at the cellular and sub-subcellular level, to detect the novel receptor tyrosine kinase protein of the invention, to localise it to particular cells and tissues and to specific subcellular locations, and to quantitate the level of expression.
  • Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detert the novel tyrosine kinase of the invention.
  • an antibody of the invention may be labelled with a detectable substance and the novel receptor tyrosine kinase of the invention may be localised in tissue based upon the presence of the detectable substance.
  • detectable substances include various enzymes, fluorescent materials, luminescent materials and radioactive materials.
  • Suitable enzymes indude horseradish peroxidase, biotin, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin
  • an example of a luminescent material includes luminol
  • suitable radioactive material include radioactive iodine I 125 , 1 1 31 or tritium.
  • Antibodies may also be coupled to electron dense substances, such as ferritin or colloidal gold, which are readily visualised by electron microscopy.
  • Radioactive labelled materials may be prepared by radiolabeling with 12 5 1 by the chloramine-T method (Greenwood et al, Biochem. J. £2:114, 1963), the lactoperoxidase method (Marchalonis et al, Biochem. J. 124:921. 1971), the Bolton-Hunter method (Bolton and Hunter, Biochem. J. 13_3_:529, 1973 and Bolton Review 18, Amersham International Limited, Buckinghamshire, England, 1977), the iodogen method (Fraker and Speck, Biochem. Biophys. Res. Commun. 8_Q_:849, 1978), the Iodo-beads method (Markwell Anal. Biochem. 125:427, 1982) or with tritium by reductive methylation (Tack et al., J. Biol. Chem. 255:8842, 1980).
  • Known coupling methods for example Wilson and Nakane, in
  • Fluorescent labelled materials may be prepared by reacting the material with umbelliferone, fluorescein, fluorescein isothiocyanate, dichlorotriazinylamine fluorescein, dansyl chloride, derivatives of rhodamine such as tetramethyl rhodamine isothiocyanate, or phycoerythrin.
  • Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specifidty for the antibody reactive against the novel tyrosine kinase of the invention.
  • a second antibody having specifidty for the antibody reactive against the novel tyrosine kinase of the invention.
  • the antibody having specificity against the novel tyrosine kinase protein of the invention is a rabbit IgG antibody
  • the second antibody may be goat anti-rabbit gamma-globulin labelled with a detectable substance as described herein.
  • the novel tyrosine kinase of the invention may be localized by radioautography.
  • the results of radioautography may be quantitated by determining the density of particles in the radioautographs by various optical methods, or by counting the grains.
  • the expression patterns found for the novel tyrosine kinase of the invention indicate that it plays unique and important roles in the determination, migration and pathfinding of axons, in axogenesis and fasciculation, development and regeneration of the neural tube, and in the regulation of spedfic cell-cell interactions during early development of the nervous system. Therefore, the above described methods for detecting nucleic acid molecules and fragments thereof and Nuk protein and parts thereof, can be used to monitor vertebrate axonal migration, fasciculation and regeneration by detecting and localizing the novel tyrosine kinase protein of the invention in migrating axons and in the migrating membrane surface of cells of the developing nervous system.
  • a substance which affects expression of Nuk protein may be assayed using the above described methods for detecting nudeic acid molecules and fragments thereof and Nuk protein and parts thereof, by comparing the pattern and level of expression of the Nuk protein or parts thereof, in the presence and absence of the substance.
  • the invention also provides methods for identifying substances which are capable of binding to the Nuk protein, or isoforms and parts thereof.
  • the methods may be used to identify ligands and natural and synthetic derivatives of such ligands, which are capable of binding to and in some cases activating the receptor tyrosine kinase protein of the invention, isoforms thereof, or part of the protein.
  • Substances which can bind with the receptor tyrosine kinase protein of the invention may be identified by reacting the novel receptor tyrosine kinase protein which is expressed in migrating vertebrate axons isoforms thereof, or part of the protein, with a substance which potentially binds to the novel receptor tyrosine kinase protein, isoforms thereof, or part of the protein such as the extracelluar domain, and assaying for substance-receptor complexes, for free substance or for non-complexed receptor tyrosine kinase protein isoforms thereof or part of the protein, or for activation of the receptor tyrosine kinase protein.
  • Conditions which permit the formation of substance-receptor protein complexes may be selected having regard to factors such as the nature and amounts of the substance and the receptor protein.
  • the substance-receptor complex, free substance or non-complexed proteins may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof.
  • antibody against the receptor protein or the substance, or a labelled receptor protein, or a labelled substance may be utilized.
  • Antibodies, receptor protein or substance may be labelled with a detectable substance as described above.
  • the receptor tyrosine kinase protein, isoforms or parts thereof, or substance used in the method of the invention may be insolubilized.
  • the receptor protein or substance may be bound to a suitable carrier.
  • suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc.
  • the carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc.
  • the insolubilized receptor tyrosine kinase protein or substance may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.
  • the receptor tyrosine kinase protein, parts thereof, or substance may also be expressed on the surface of a cell using the methods described herein.
  • the above mentioned methods of the invention may be used to identify ligands which bind with and activate the novel receptor tyrosine kinase protein of the invention thereby affecting signalling pathways, particularly those involved in neuronal development and axonal migration and regeneration. Identification and isolation of such a Nuk protein ligand will permit studies of the role of the ligand in the developmental regulation of axonogenesis and neural regeneration, and permit the development of substances which affect these roles, such as functional or non-functional analogues of the ligand. It will be appredated that such substances will be useful as pharmaceuticals to modulate axonogenesis, nerve cell interactions and regeneration to treat conditions such as neurodegenerative diseases and cases of nerve injury.
  • Ligands which bind to and activate the novel receptor tyrosine kinase protein of the invention may be identified by assaying for protein tyrosine kinase activity i.e. by assaying for phosphorylation of the tyrosine residues of the novel receptor tyrosine kinase protein.
  • Receptor tyrosine kinase protein activity may be assayed using known techniques such as those using anti-phosphotyrosine antibodies and labelled phosphorous. For example, immunoblots of the complexes may be analyzed by autoradiography (32p-labelled samples) or may be blocked and probed with antiphosphotyrosine antibodies as described in Koch, CA. et al (1989) Mol. Cell. Biol. 9, 4131-4140.
  • the ligands for many receptor tyrosine kinase proteins are cell-bound, either as they are associated with the cell surface via heparin and hepatocyte growth factor or because they are transmembrane proteins (Lyman et al. 1993, supra).
  • Ligands for receptor tyrosine kinases of the EphlElklEck subfamily of receptor tyrosine kinases may require cell-to-cell contact to activate the receptor.
  • Membrane attachment of the ligand could facilitate ligand dimerization or clustering, or both, which may promote receptor multimerization and activation.
  • a ligand for Nuk protein may have a cell-bound form.
  • a cell-bound ligand may be identified by reacting the receptor tyrosine kinase protein of the invention, an isoform or a part thereof with a cell suspeded of expressing the ligand on the surface of the cell following the procedures generally described in Lyman et al., 1993, (Cell 75:1157-1167).
  • the invention provides a method for identifying cells expressing a surface bound ligand of Nuk protein and for specifically selecting for such cells.
  • a cDNA encoding a ligand for Nuk protein may be doned by first constructing a fusion protein.
  • the fusion protein may consist of the extracelluar domain of Nuk protein (amino acids 26 to 548, SEQ ID NO: 2 and Figure 2).
  • the fusion protein may be expressed and used as a probe to examine cells or cell lines (e.g. neuroblastoma or neuroepitheliomia cell lines) for their capacity to bind the extracellular domain of Nuk protein (determined by flow cytometry).
  • the identification of cells and cell lines that bind the extracellular domain may be facilitated by incorporating in the fusion protein a sequence encoding a marker protein for example, the Fc portion of human IgG which may be detected with labelled anti-human IgG antibodies.
  • a marker protein for example, the Fc portion of human IgG which may be detected with labelled anti-human IgG antibodies.
  • Cells or cell lines which bind the extracellular domain are presumed to express a cell-bound form of the ligand.
  • a cDNA expression library is constructed, following known techniques, using mRNA from the cells /cell lines which have been identified as binding the fusion protein containing the extracellular domain of Nuk protein. cDNAs are then transfected into host cells (e.g. COS cells and see discussion herein re host cells) which are then screened for their capacity to bind the extracellular domain of Nuk protein. Individual clones whid are capable of binding the extracellular domain of Nuk protein are identified and the cDNAs are sequenced. The cDNAs may be used as hybridization probes to isolate genomic DNA encoding the ligand.
  • host cells e.g. COS cells and see discussion herein re host cells
  • the invention also contemplates a method for assaying for an agonist or antagonist of the binding of the novel receptor tyrosine kinase of the invention with a substance which is capable of binding with the novel tyrosine kinase protein, preferably a ligand.
  • the agonist or antagonist may be an endogenous physiological substance or it may be a natural or synthetic drug.
  • Substances which are capable of binding with the Nuk protein and preferably ligands, including cell-bound ligands may be identified using the methods set forth herein.
  • Substances which bind to other receptor tyrosine kinases of the EphlElklEck subfamily may also be used in this assay.
  • the substance may be LERK-2, a binding protein for the receptor- tyrosine kinase ELK (Fletcher, F.A. et al., Oncogene (1994), 9, 3241-3247), or the cell-bound ligands B61 (also known as EFL-1), EHK1-L (also known as EFL-2) and ELK-L (also known as EFL-3)(Davis, S. et al., Sdence Vol. 266, ⁇ .816, Nov. 4, 1994).
  • ELK receptor- tyrosine kinase
  • a method which comprises providing a known concentration of the novel receptor tyrosine kinase protein of the invention, incubating the protein with a ligand which can bind to and activate the protein, and a suspected agonist or antagonist under conditions which permit the formation of substance-receptor protein complexes, and assaying for substance-receptor protein complexes, for free substance, for non-complexed proteins, or for activation of the receptor tyrosine kinase protein.
  • the agonists and antagonists that can be assayed using the methods of the invention may act on one or more of the binding sites on the receptor tyrosine kinase or the ligand, including agonist binding sites, competitive antagonist binding sites, non-competitive antagonist binding sites or allosteric sites.
  • the invention also makes it possible to screen for antagonists that inhibit the effects of an agonist of the interaction of Nuk protein with a Nuk protein ligand.
  • the invention may be used to assay for a substance that competes for the same ligand binding site of the novel receptor tyrosine kinase protein of the invention.
  • the invention further contemplates a method for identifying a substance which is capable of binding to an activated receptor tyrosine kinase protein of the invention or an isoform or part of the activated protein, comprising reacting an activated receptor tyrosine kinase protein of the invention, or an isoform, or part of the protein, with at least one substance which potentially can bind with the receptor tyrosine kinase protein, isoform or part of the protein, under conditions which permit the formation of substance-receptor kinase protein complexes, and assaying for substance-receptor kinase protein complexes, for free substance, for non-complexed receptor kinase proteins, or for phosphorylation of the substance.
  • An activated receptor tyrosine kinase protein of the invention, or isoform or part thereof may be prepared by binding of a ligand to the extracellular domain of a receptor tyrosine kinase protein of the invention which results in activation of the catalytic domain.
  • a ligand may be identified using the methods hereinbefore described.
  • An activated receptor or part thereof may also be prepared using the methods described for example in Reedijk et al. The EMBO Journal, 11(4):1365, 1992 for producing a tyrosine phosphorylated receptor or part thereof.
  • substance-receptor protein complexes Conditions which permit the formation of substance-receptor protein complexes may be seleded having regard to factors such as the nature and amounts of the substance and the receptor protein.
  • the substance-receptor complex, free substance or non-complexed proteins may be isolated by conventional isolation techniques described above. Phosphorylation of the substance may be determined using for example, labelled phosphorous as described above.
  • intracellular ligands such as Src homology region 2 (SH2)-containing proteins which are capable of binding to a phosphorylated activated receptor tyrosine kinase protein of the invention may be identified.
  • SH2-containing proteins refers to proteins containing a Src homology region 2 which is a noncatalytic domain of -100 amino acids which was originally identified in the Vfps and Vsrc cytoplasmic tyrosine kinases by virtue of its effects on both catalytic activity and substrate phosphorylation (T. Pawson, Oncogene 3, 491 (1988) and I. Sadowski et al., Mol. Cell. Biol. 6, 4396 (1986)).
  • SH2-containing proteins may function downstream of the Nuk signalling pathway by binding to the activated receptor protein.
  • the cytoplasmic tyrosine kinases of the Src family may bind via their SH2 domains to the activated Nuk receptor protein thereby regulating cellular processes particularly in the nervous system.
  • Intracellular ligands which may be phosphorylated by the novel receptor tyrosine kinase of the invention may also be identified using the method of the invention.
  • SH2-domains of cytoplasmic signalling proteins have been found to bind to the phosphorylated receptor tyrosine kinase protein of the invention.
  • the SH2 domains of p21 «s GTPase-activating protein (GAP), Src, and phosphoinositide-spedfic phospholipase C (PLC ⁇ ) have been found to bind Nuk protein.
  • GAP GTPase-activating protein
  • Src Src
  • PLC ⁇ phosphoinositide-spedfic phospholipase C
  • the SH-2 domain binding site on the Nuk protein is a conserved tyrosine containing region which is located adjacent to the membrane and it corresponds to amino acids 600 to 618 as shown in SEQ. ID. NO:2 in the Sequence Listing.
  • the invention also contemplates a method for assaying for an agonist or antagonist of the binding of an activated receptor tyrosine kinase of the invention, or a portion thereof with an SH2 domain of an intracellular ligand.
  • the agonist or antagonist may be an endogenous physiological substance or it may be a natural or synthetic drug.
  • the activated receptor may be prepared as described herein or a portion of the ligand comprising the amino acid sequence 600 to 618 as shown in SEQ. ID. NO:2 in the Sequence Listing may be used in this method of the invention.
  • SH2 domains of intracellular ligands indude the SH2 domains of GAP, Src, and PLC ⁇ . It will be appreciated that the entire intracellular ligand may be used in this method.
  • COS cells express large amounts of Nuk protein. Accordingly, COS cells may be used to identify in vivo, intracellular proteins or ligands which bind to the Nuk protein.
  • the invention further provides a method for assaying for a substance that affects axonal migration, neural development, nerve cell interactions and nerve regeneration comprising administering to a non-human animal or to a tissue of an animal, a substance suspected of affecting axonal migration, and detecting, and optionally quantitating, the novel receptor tyrosine kinase of the invention in the non-human animal or tissue.
  • the method may be used to assay for a substance that affects axonal migration during embryogenesis.
  • the novel receptor tyrosine kinase of the invention may be quantitated using the methods described herein.
  • the method may be used to assay for a substance that affects axonal migration in nerve regeneration, comprising administering a substance suspected of affecting axonal migration to a non-human animal having an injured peripheral nervous system and detecting, and optionally quantitating, the novel receptor tyrosine kinase of the invention in the non-human animal.
  • non-human animals having an injured peripheral nervous system include animals having damaged axons, such as axotomized facial neurons (Sendtner et al.
  • neurodegenerative conditions for example, the MPTP model as described in Langston J.W. et al., Symposium of Current Concepts and Controversies in Parkinson's Disease, Montebello, Quebec, Canada, 1983 and Tatton W.G. et al., Can. J. Neurol. Sci. 1992, 19
  • traumatic and non-traumatic peripheral nerve damage for example, animal stroke models such as the one described in MacMillan et al. Brain Research 151:353-368 (1978)).
  • Substances which are capable of binding to the Nuk protein of the invention or parts thereof, particularly ligands, and agonists and antagonists of the binding of ligands and Nuk protein, identified by the methods of the invention, may be used for stimulating or inhibiting neuronal development, regeneration and axonal migration.
  • the ligands, agonists and antagonists may accordingly be used to stimulate or inhibit neuronal development, regeneration and axonal migration associated with neurodegenerative conditions and conditions involving trauma and injury to the nervous system, for example Alzheimer's disease, Parkinson's disease, Huntington's disease, demylinating diseases, such as multiple sclerosis, amyotrophic lateral sclerosis, bacterial and viral infections of the nervous system, deficiency diseases, such as Wernicke's disease and nutritional poly neuropathy, progressive supranuclear palsy, Shy Drager's syndrome, multistem degeneration and olivo ponto cerebellar atrophy, peripheral nerve damage, trauma and ischemia resulting from stroke.
  • demylinating diseases such as multiple sclerosis, amyotrophic lateral sclerosis, bacterial and viral infections of the nervous system
  • deficiency diseases such as Wernicke's disease and nutritional poly neuropathy, progressive supranuclear palsy, Shy Drager's syndrome, multistem degeneration and olivo pont
  • the invention also provides methods for studying the function of the Nuk protein.
  • Cells, tissues, and non-human animals lacking in Nuk expression or partially lacking in Nuk expression may be developed using recombinant molecules of the invention having specific deletion or insertion mutations in the Nuk gene.
  • the extracellular domain or parts thereof such as the FN m and Ig domains; the transmembrane region or parts thereof; the tyrosine kinase domain or parts thereof, such as the ATP binding site and; the carboxy terminal tail may be deleted.
  • a recombinant molecule may be used to inactivate or alter the endogenous gene by homologous recombination, and thereby create a Nuk defi ⁇ ent cell, tissue or animal.
  • Null alleles may be generated in cells, such as embryonic stem cells by deletion mutation.
  • a recombinant Nuk gene may also be engineered to contain an insertion mutation which inactivates Nuk.
  • Such a construct may then be introduced into a cell, such as an embryonic stem cell, by a technique such as transfection, electroporation, injection etc.
  • Cells lacking an intart Nuk gene may then be identified, for example by Southern blotting, Northern Blotting or by assaying for expression of Nuk protein using the methods described herein. Such cells may then be fused to embryonic stem cells to generate transgenic non-human animals deficient in Nuk.
  • Germline transmission of the mutation may be achieved, for example, by aggregating the embryonic stem cells with early stage embryos, such as 8 cell embryos, in vitro; transferring the resulting blastocysts into recipient females and; generating germline transmission of the resulting aggregation chimeras.
  • Such a mutant animal may be used to define spedfic nerve cell populations, developmental patterns of axonogenesis, neural tube formation and nerve regeneration and in vivo processes, normally dependent on Nuk expression.
  • the present inventors have generated a loss of function deletion mutation in Nuk, designated Nu in mouse embryonic stem cells, and have achieved germline transmission of this null allele.
  • Nuk mutation was obtained by deletion of exon 2, corresponding to codons 29 to 50, as shown in Figure 9.
  • Adult animals homozygous for the mutation did not produce any Nuk protein.
  • a second targeted mutation, designated Nuk 2 was generated in the Nuk gene as shown in Figure 10 using the pPNT-LOX-Nuk 2 gene trap vector to delete the GXGXXG ATP binding region of the kinase domain (amino adds 623-707, SEQ ID NO:2 and Figure 2) and to create a Nuk-lac Z fusion receptor.
  • Chimeric animals expressing Nu k 2 were prepared. Animals generated with the Nuk 2 mutation provided Nuk 2 expressing cells staining for ⁇ -galactosidase activity, providing a convenient marker for Nuk -positive cells in both heterozygous and homozygous backgrounds as deteded by a blue/ green colour as shown in Figures 11 and 12.
  • the invention also provides methods for preparing cells, tissues, and non-human animals lacking in Nuk expression or partially lacking in Nuk expression, and deficient in the expression of other genes.
  • an animal may be generated which is defident in Nuk and another tyrosine kinase of the EphlElklEck subfamily. Such animals could be used to determine how the members of the EphlElklEck subfamily co-operate in embryonic development, particularly development of the nervous system.
  • an animal lacking or partially lacking Nuk expression and Sek-4 expression may be generated to determine how the receptor tyrosine kinases co-operate in the segmental patterning of the hindbrain.
  • mice can also be generated to study the interaction of Nuk protein and other proteins such as the Src-family of cytoplasmic tyrosine kinases.
  • an animal may be generated which lacks or partially lacks Nuk expression, and expression of one or more Src family tyrosine kinases induding Src, Fyn, and/or Yes.
  • DNAs generated in this study were subcloned into either the pGEM7 (Promega) or pCRTI (Invitrogen) plasmid vectors prior to double stranded sequencing using the Sequenase system (United States Biochemical Corporation). Sequencing reactions were primed using either the standard forward and reverse primers or custom oligonudeotides synthesized in house on a Pharmacia Gene Assembler Plus.
  • Chromosome mapping was performed by probing Pst I restriction endonulcease digested DNAs from 28 different recombinant inbred mouse strains at high stringency with a 1.0 Kb EcoRI fragment of the ⁇ Ql Nuk cDNA. Generation of antibodies
  • a trpE-Nuk fusion protein was created by subcloning a NcoI-BamHI fragment of ⁇ Ql containing the carboxy terminal 94 Nuk codons plus 170 additional nucleotides of 3' untranslated sequences into the bacterial expression vector pATHl.
  • a GST-Nuk fusion protein containing Nuk amino acids 601-994 was also expressed in bacteria.
  • the trpE-Nuk fusion protein was purified from induced bacterial cultures by SDS-PAGE and used to immunize rabbits. Resulting trpE-Nuk antiserum was affinity-purified by binding to immobilized glutathione agarose purified GST-Nuk. Immunoprecipitation. in vitro kinase. and western blotting
  • the embryo lysates were clarified by centrifugation at 12,000 x rpm for 10 min at 4°C. The cleared supernatant was put on ice and an aliquot was used for protein assay (BCA protein assay, PIERCE).
  • BCA protein assay PIERCE
  • the immunoprecipitates were collected by centrifugation at 12,000 x rpm for 30sec and washed three times with ice cold HNTG buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, and 1 mM Na 3 V0 ).
  • the washed immunoprecipitates were incubated at room temperature (RT) for 15 minutes in 20 ml of kinase buffer (20 mM HEPES, pH7.5, 25 mM MgCl 2 , 4 mM MnCl 2 , and 0.1 mM Na 3 V0 ) containing 10 ⁇ Ci of ⁇ 32P-ATP (Dupont; 3000 Ci/mmole-i).
  • the immune complex kinase reaction products were denatured at 100 ⁇ C for 5 min in SDS sample buffer and separated by SDS-polyacrylamide gel electrophoresis. Gels were fixed in acetic add, submerged in 1 M KOH at 55°C for 45 min to remove phosphoserine and phosphothreonine, refixed, dried, and then exposed to Kodak XAR film.
  • the washed immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis and electro-transferred to a nylon filter using a semi-dry protein blotting apparatus.
  • the filter was blocked overnight at 4°C in PBS containing 5% dry milk prior to incubation in the same solution containing 1 ⁇ g/ml anti-Nuk antibodies for lhr at room temperature (RT).
  • the filter was washed at RT 3 X10 min with TBSN (20 mM TrisHCl, pH 7.5, 150 mM NaCl, and 0.05% NP-40).
  • the immunohistochemical detection used in this study was based on the Vectastain ABC Elite-peroxidase system and Vectastain biotin/avidin blocking reagents (Vector Laboratories).
  • the specificity of the anti-Nuk antibody staining was confirmed by a variety of control experiments induding those in which the primary antibody was either omitted or preabsorbed with a trpE-Nuk peptide.
  • other antibodies including a monoclonal antibody raised against the 160 kD subunit of Neurofilament (anti-NF; AMAC Inc.) and a rabbit polyclonal antibody raised against the murine Engrailed (En) proteins were used to control for the specificity of the immunohistochemistry. All observations reported here have been derived from a number of independent experiments all of which gave similar results. The total number of anti-Nuk stained embryos observed at each stage was from 40 to over 100.
  • Embryos used in this study were obtained from natural matings of CD1 mice and at the required stages of development were dissected and membranes were removed in ice cold 0.1 M phosphate buffer (pH 7.4). Embryos were then fixed with occasional mixing for 2 h on ice in fresh 4% paraformaldhyde in 0.1 M phosphate buffer. In some instances 0.2 to 1% glutaraldhyde was also added to the fixative.
  • the fixative was washed out overnight in 0.1 M phosphate buffer at 4°C prior to gently dehydrating the embryos in a methanol/ phosphate buffered saline (PBS) series (15%/85%; 30/70; 50/50; 75/25% H 2 0; 100% methanol) on ice for 30 minutes each step. Once in 100% methanol the embryos can be stored at -20°C for at least 2 months. Prior to rehydration and further manipulation, the embryos were transferred into 80% methanol /20% H 2 0 2 for 4-6 h at RT to bleach embryos and inactivate endogenous peroxidases.
  • PBS methanol/ phosphate buffered saline
  • the embryos were rehydrated on ice for 30 minutes each with occasional mixing in 75% methanol/25% H 2 0, 50% methanol/50% PBS, 30% methanol/70% PBS, 15% methanol/85% PBS, and 100% PBS + 0.01% Triton X-100.
  • the embryos were then gently mixed on a Nutator at RT 2 x 1 h in PBSMT (2% Dry Milk Powder, 0.01% Triton X-100 in PBS). At this step a small number of embryos were placed at 4°C in PBSMT for preabsorbtion of the biotinylated secondary antibodies as described by the manufacturer (Vector Laboratories).
  • the embryos were then blocked overnight at 4 ⁇ C on Nutator using the Vedastain ABC Elite and blocking kits in PBSMT+3% normal goat serum (NGS) + 10% avidin blocking reagent.
  • the avidin was then washed out in PBSMT 4 x 1 hour at 4°C followed by 2 x 1 hour at RT.
  • the blocked embryos were then incubated on a Nutator for at least 16 h at 4 ⁇ C with affinity-purified anti-Nuk antibodies (0.5 to 1.0 mg/ml) in PBSMT containing 3% normal goat serum and 10% biotin blocking agent. Unbound primary antibodies and biotin were washed out in PBSMT 4 x 1 hour at 4 ⁇ C followed by 2 x 1 hour at RT.
  • the preabsorbed biotinylated secondary antibodies were then added to the embryos and incubation was carried out overnight at 4°C on a Nutator.
  • the secondary antibodies were washed out as described above and the Vectastain ABC elite avidin-biotin-HRP reagent in PBSMT+3% NGS was added and allowed to incubate overnight at 4°C on a Nutator.
  • the ABC elite avidin-biotin-HRP reagent was washed out as described above ending with a final wash in PBT (0.2% BSA, 0.01% Triton X-100 in PBS) at RT.
  • FIRP detection embryos were incubated in 0.3 mg/ml diaminobenzidine (DAB) in PBT at RT for at least 20 minutes. H 2 0 2 was added to 0.03% and the embryos were incubated at RT under a dissecting microscope until color density was suffident, usually about 1-10 minutes. The color of HRP-DAB reaction product can be changed from an orange to dark purple color by the addition of NiCl 2 to 0.5%. After staining, embryos were washed in PBT and dehydrated through a ethanol/PBS series: 30/70, 50/50, 80/20, 100% ethanol for 30 minutes each. For light microscopy, embryos were cleared in benzyl alcohokbenzyl benzoate (1:2).
  • DAB diaminobenzidine
  • the xylene was exchanged with many changes of melted paraffin for 2 to 4 hours at 60 ⁇ C prior to embedding. Embryos were sectioned at 4 to 6 ⁇ m and placed on slides freshly subbed in 1% aminopropyltriethoxysilane. Sections were placed on a slide dryer for 1 hour, allowed to dry overnight at RT, and then partially melted at 55"C for 20 min. The antibody staining was performed exactly as described in the
  • Anti-Nuk antibodies were used at 4 to 8 ⁇ g/ml.
  • Nuk+peptide control 8 ⁇ g/ml of trpE-Nuk fusion protein was added to the anti-Nuk antibodies and preincubated for 1 hour before adding to the sections.
  • Anti-NF monoclonal antibodies were used at 4 ⁇ g/ml.
  • the HRP reaction was carried out in DAB only. These sections were counterstained in hematoxylin prior to mounting with coverslips. In other sections, the HRP reaction was carried out in DAB+NiCl 2 to produce a darker higher contrast product for black and white photography. These sections were not counterstained prior to mounting.
  • the Nuk Ig-like domain When compared to the Ig-like domains found in other receptor tyrosine kinases (O'Bryan et al, Mol. Cell. Biol. 11:5016, 1991), the Nuk Ig-like domain was found to contain a number of conserved residues (overlines. Figure 2). A repeat involving the Nuk Ig-like domain and residues 239 to 268 is apparent (underlines. Figure 2). Although significantly shorter than a normal Ig domain, residues involved in the Nuk-specific repeat correspond to conserved residues often found in Ig-like domains (residues that are both overlined and underlined in Figure 2). Following the transmembrane domain, the Nuk cytoplasmic region contains a tyrosine kinase catalytic domain (brackets. Figure 2) and a carboxy-terminal tail of 106 residues.
  • the cartoon in Figure 2 shows the location of the various domains.
  • the carboxy-terminal region used to raise the anti-Nuk antibodies is indicated in Figure 2.
  • the Nuk protein extracelluar domain is composed of an Ig-like domain and two FN in repeats.
  • the Nuk protein extracelluar domain also contains 20 cysteines whose position is conserved in the Eph family (Lhotak et al, Mol. Cell Biol. 11:2496-2502, 1991).
  • a hydrophobic transmembrane domain divides the Nuk protein into approximately two halves, a 548 amino add extracelluar region and a 419 amino acid cytoplasmic region which contains a tyrosine kinase catalytic domain.
  • Nuk protein sequence was compared to other known members of the Eph family. Nuk was found to be most highly related to the full length amino acid sequence of chicken Cek5 (96% identity; Pasquale, Cell Regulation 2:523-534, 1991) and to short PCR products of mRNA from rats (Tyro 5; Lai and Lemke, Neuron 6:691-704, 1991) and humans (Erk; Chan and Watt, Oncogene 6:1057-1061 1991).
  • the dose identity between Nuk and Cek5 suggest they represent the mammalian and avian orthologs of the same progenitor gene. The absence of full length cDNAs for Tyro 5 and Erk precludes the determination of whether these sequences correspond to the same or a dosely related but different gene.
  • the chromosomal location of Nuk was determined by probing for restriction fragment length polymorphisms (RFLPs) in the DNA of a number of recombinant inbred mouse strains derived from matings between AKR/J and DBA/2J mice (B.A. Taylor, personal communication). The Nuk locus mapped to the distal end of mouse chromosome 4 near the ahd-1 mutation.
  • RFLPs restriction fragment length polymorphisms
  • Nuk protein was immunoprecipitated from 10.5, 12.5, and 14.5 day mouse embryo protein lysates and then incubated in the presence of [ ⁇ pjATP.
  • the expression and tyrosine kinase activity of Nuk in the embryos is shown in Figure 3.
  • Anti-Nuk protein antibodies immunoprecipitated a protein-tyrosine kinase of 135 kD, as shown in Figure 3A.
  • a highly phosphorylated protein with a relative mobility of 135 kD was detected in protein extracts of 10.5 day (lane 3, Figure 3A), 12.5 day (lane 4, Figure 3A), and 14.5 day (lane 5, Figure 3A) embryos.
  • the mobility of this protein is consistent with that reported for other Eph family members including Eck, Elk, and Cek5 (Lindberg et al, Mol Cell Biol. 10:6316-6324, 1990; Lhotak et al, Mol Cell Biol. 11:2496-2502, 1991; Pasquale et al, ⁇ . Neurosci.
  • Figure 4 Nuk protein localization in whole-mount and paraffin sections of 7.5 to 10.5 day mouse embryos is shown in Figure 4, comprising whole-mount preparations showing Nuk mRNA (Figure 4A) and protein ( Figures 4B to 4E) at early postimplantation stages of embryonic development. Unless otherwise stated, in Figure 4, dorsal is left and anterior is up.
  • Figure 4A represents whole-mount mRNA studies which detected Nuk transcripts enriched in the neural ectoderm of an embryo at 7.5 days development (note dorsal surface is up). By the 6 somite stage (8 days) Nuk protein is detected as dark orange horseradishperoxidase (HRP) staining as shown in Figure 4B.
  • HRP horseradishperoxidase
  • FIG. 4B points to rhombomeres r2, r3 that express high levels of Nuk protein.
  • the sinus venosis of the developing circulatory system also stains positive for Nuk.
  • Figure 4C shows an embryo at 12 somite stage (8.75 days) and reveals elevated levels of Nuk protein in specific regions of the anterior neural tube.
  • the most anterior structures immunoreactive for Nuk include the ventral diencephalon followed by the ventral mesencephalon/midbrain.
  • Rhombomeres r2, r3, and r5 of the hindbrain are also immunoreactive for Nuk protein.
  • the arrow in Figure 4C points to rhombomere 5.
  • the expression of Nuk was confirmed by performing mRNA in situs on similar staged embryos.
  • Figure 4D shows 24 somite (9.5 day) stage embryos immunoreaded with either anti-Nuk protein antibodies (left) or, as a control, anti-Nuk protein antibodies that were preincubated with a trpE-Nuk fusion protein (right). Nuk protein is most abundant in the ventral midbrain, diencephalon, and optic stalk. Along the spinal cord, high levels of Nuk protein is detected at the dorsal surface of the neural tube (see Figure 5 for more detail). Note the absence of specific staining in the control embryo.
  • Figure 4E shows whole-mount immunolocalization of Nuk protein in the developing brain of a 9.5 day embryo in greater detail. Nuk protein is most highly expressed in the ventral midbrain encompassing the flexure region.
  • Nuk protein is also detected in the ventral diencephalon, optic chiasm, optic stalk, retinal cells (out of focus), and basal telencephalon. At this stage, Nuk protein is still detected in the hindbrain and is localized to the floorplate (arrow in Figure 4E points to ventral region of rhombomere 2). Note the small patch of Nuk protein now detectable in the lateral region of rhombomere 4 just anterior of the otic vesicle.
  • Figures 4F to 4J show immunohistochemical detection of Nuk protein in paraffin sections of 9.5 and 10.5 day mouse embryos.
  • Figures 4F and 4G show serial transverse sections of a 9.5 day embryo immunoreacted with anti-Nuk antibodies which detect Nuk protein (brown stain) in the ventral midbrain.
  • Nuk protein is localized to all layers of the neural tube including the proliferative ventricular zone, the internal mantel layer, and the outer marginal layer which is adjacent to the surrounding mesenchyme.
  • Nuk protein is detected only in the ventral aspect of the midbrain and its limit of expression marks a spedfic morphological bulge/constriction of the neural tube that separates ventral from dorsal components (arrowhead).
  • Figure 4H shows a sagittal section along the midline of a 10.5 day embryo showing Nuk protein (dark stain) concentrated in ventral regions of the midbrain and in the optic chiasm (asterisk) and footplate of the hindbrain (thick arrow). The orientation of this embryo is opposite to the one shown in Figure 4E. Note the appearance of a morphological constriction (arrowhead) which separates the midbrain from the diencephalon. Since this is a section along the midline, the infundibulium or ventral most region of the diencephalon is exposed between the midbrain and optic chiasm. This region of the diencephalon does not express Nuk.
  • Figures 41 and 4J show adjacent transverse sections of a 10.5 day embryo immunoreacted with either anti-Nuk protein antibodies (41) or anti-Nuk antibodies preincubated with a trpE-Nuk peptide (4J), and illustrate the specificity of the immunohistochemistry. Staining of Nuk protein at 10.5 days persists in the ventral midbrain and is also detected at lower levels in the floorplate of the hindbrain (arrowhead).
  • the scale bars shown in Figure 4 represent the following measurements: (4A), 100 ⁇ m; (4B and 4C), 120 ⁇ ; (4E, 4F, 4G, 41, 4J), 150 ⁇ m; (3H), 300 ⁇ m.
  • the immunohistochemical reaction product observed for the anti-Nuk antibodies often appears as if the Nuk protein is localized to specific surfaces of the plasma membrane.
  • One clear example of this is at the dorsal surface of the posterior neural tube which contains elevated amounts of the Nuk protein as early as 9.5 days of development (see Figure 4D).
  • the subcellular localization of Nuk protein is shown in more detail in Figure 5.
  • Figures 5 A and 5B are adjacent transverse sections of an 11.5 day embryo at the level of the caudal/posterior spinal cord immimoreacted with either anti-Nuk antibodies (5A) or anti-Nuk antibodies preincubated with a trpE-Nuk peptide (5B).
  • Nuk protein is concentrated in the dorsal region of the neural tube along the basement membrane (arrow). This localization of Nuk protein is observed as early as 9.5 days of development in whole-mount preparations (see Figure 4D).
  • Antibodies to Neurofilament (anti-NF) and Engrailed (anti-En) proteins did not stain the basement membrane demonstrating the specificity of the anti-Nuk antibodies.
  • Figures 5C and 5D show transmission electron microscopy (EM) immunolocalization of Nuk protein in ventral midbrain cells of a 9.5 day embryo. Immunoperoxidase anti-Nuk stained whole-mount embryos were ultrathin sectioned and observed under EM.
  • Figure 5C shows that, at the proliferative ventricular zone (VZ), Nuk-positive signals are concentrated at sites of cell-cell contact (arrows). Notice that Nuk protein immunostaining is often localized on the membranes of both cells that are making contact. At this stage of development there is no obvious ultrastructural differences between neuronal and glial cell types. Therefore, it cannot be distinguished if Nuk protein is associated with neurons, glial, or both cell types. Note the condensed d romatin evident in the nudeus of a cell that is in late prophase (asterisk).
  • Figure 5D shows that cells within the postmitotic mantal region of the neural tube also exhibit Nuk protein localization at sites of cell-cell contact.
  • the photomicrograph shows a site of contact between two cells labelled strongly for Nuk protein (filled arrow). Note that other sites of cell-cell contad do not contain detectable amounts of Nuk protein (open arrows).
  • the scale bars in Figure 5 represent the following measurements: (A and B) 100 ⁇ m; (C), 2 ⁇ m; (D), 1 ⁇ m.
  • Nuk localization in pioneer cranial PNS axons and an early CNS axon tract and in spinal axons Axonogenesis in mouse embryos commences at approximately 10 days of development when neurons associated with both the central and peripheral nervous systems extend axon projections toward their targets.
  • Anti-Nuk antibody staining of 10 to 12 day embryos revealed that Nuk protein is highly concentrated within most if not all early axonal projections of the PNS and to at least one early pathway of the CNS.
  • Figure 6 A is a photomicrograph of the head of an 11 day anti-Nuk whole-mount immunostained embryo with one of the pair of oculomotor nerve fibers in focus showing strong labelling of Nuk protein (filled arrow).
  • Figure 6B A clearer view of Nuk protein staining in the oculomotor axons fibers can be obtained when the whole-mount staged embryo was filleted down the midline to minimize tissue thickness (Figure 6B). This view shows that the Nuk protein positive oculomotor in axons exit the neural tube from the ventral aspect of the midbrain.
  • Anti-Nuk stained frontal section shows that these axons originate from the Nuk-expressing cells in the ventral midbrain, at a region that is consistent with the position of the oculomotor nuclei ( Figure 6E).
  • Anti-Nuk antibodies also label an early pathway of the CNS (open arrow in Figure 6).
  • Figures 6C to 6F show paraffin section immunohistochemistry of 10.5 to 11 day mouse embryos. Adjacent transverse sections bisecting the pair of oculomotor nerve fibers were immunostained with either anti-Nuk (6C) or anti-Neurofilament (6D) antibodies. The arrows in both Figures 6C and 6D point to the darkly stained axon bundles of the two oculomotor nerves. To verify the specificity of the immunohistochemistry, additional control experiments were performed in which the anti-Nuk antibodies were either omitted, preincubated with a trpE-Nuk peptide, or substituted with antibodies directed against the Engrailed (anti-En) nuclear homeodomain proteins.
  • Figure 6E is a frontal section immunostained with anti-Nuk antibodies which label the ventral midbrain and oculomotor axon fibers as they exit the neural tube (filled arrow) and extend (open arrow) towards their target tissue, the pre-optic musde mass.
  • Figure 6F shows a sagittal section immunostained with anti-Nuk antibodies which label the oculomoter axon fascicule as it enters the pre-optic muscle mass (open arrow).
  • This section also shows the expression of Nuk protein in the developing retinal cells (r). The orientation of this section is the same as Figure 4H.
  • the scale bar for Figures 6 (A) is 180 ⁇ m; (B) 120 ⁇ m ; and (C-F) 100 ⁇ m.
  • Nuk protein is detected in the trigeminal (V) and facial (VII) nerve fibers in 10.5 day embryos ( Figures. 7A to 7C).
  • the vagus (X) parasympathetic autonomic nerve, and the accessory (XI) and hypoglossal (XII) somatomotor nerves also contain localized Nuk protein ( Figures. 7D and 7E).
  • these fibers enter a common region, the cardiac/pulmonary plexus, where they then elongate to their targets such as the cardiac muscle and other visceral organs (vagus) or the upper torso (accessory) and tongue (hypoglossal). Nuk protein localization in these cranial axons is very transient and is not detected after 12.5 days development.
  • the embryo in Figure 6A and B also exhibits specific anti-Nuk labelling in the developing CNS of a connection between the telencephalon and the midbrain.
  • Information describing the naming and position of early tracts in the developing mammalian forebrain is sparce.
  • the location of Nuk immunoreactivity is consistent with the location of axonal projections originating from the ventral midbrain tegmentum such as those of the red nuclei or the reticular activating system (Carpenter, 1985, Core Text on Neuroanatomy, Baltimore: Williams & Wilkins).
  • this pathway may correspond to the telencephalic/supraoptic tract described in zebra (Chitnis and Kuwanda, J. Neurosci. 10, 1892-1905, 1990; Wilson et al., (1990) Development, 108, 121-143, 1990). Closer definition of the origin and termination sites as well double labelling with other antibody probes should help determine the identity of this Nuk-positive tract
  • Figure 7A is a 10.5 day whole-mount embryo immunostained with anti-Nuk antibodies which label the trigeminal nerve V and facial nerves VII.
  • Figures 7B and 7C show transverse sections of a 10.5 day embryo showing Nuk-positive trigeminal nerve V axon fascicules labelled with the anti-Nuk antibodies. Note that Nuk protein is also present throughout the caudal hindbrain region of the neural tube.
  • Figures 7D and E are 11.5 day anti-Nuk whole-mounts showing localization of Nuk protein in the vagus nerve (X) associated fibers as they pathfind to their target visceral organs (curved open arrows).
  • cranial axons induding the accessory (XI) and hypoglossal (XH) nerve fibers are immunoreactive for Nuk protein. These cranial nerves initially extend to the plexus region (open arrow in D) before they pathfind to the heart and other target tissues.
  • Figures 7F to 7L show Nuk protein localization in spinal nerves of 10 to 12 day embryos.
  • Figure 7F is a whole-mount 10 day embryo showing Nuk protein concentrated within the earliest spinal nerve fibers exiting the neural tube (arrows).
  • Figure 7G shows a slightly later stage embryo from that shown in Figure 7F. Notice that the nerve fibers exiting the neural tube have thickened due to fasciculation of additional axons.
  • the insert is a dose-up of a single spinal nerve showing Nuk protein is localized throughout its length and can be observed at the leading tips of the growth cones.
  • Figures 7H and 71 show transverse sections bisecting the rostral spinal cord of an 11 day embryo demonstrating that the darkly stained Nuk-positive fibers shown in Figures 7F and 7G are ventral motor axons (open arrows). At this stage, the accesory nerve also stains positive for Nuk protein as evident by the strong labelling of the axon fibers (filled arrows). The uniform expression of Nuk throughout the spinal region of the neural tube is apparent in these sections.
  • Figures 7J and 7K show both sides of an 11 day whole-mount embryo demonstrating that Nuk protein is localized within the spinal motor nerves as they elongate to the plexus regions (open arrows in Figure 7J). Between the motor fibers, the appearance of Nuk-positive DRG cell bodies and axons can be observed in this embryo (filled arrows in Figure 7K). Note that the Nuk-positive DRG axons are more apparent in the posterior/caudal segments of the spinal cord.
  • Figure 7L shows a close-up of a 12 day whole-mount embryo immunostained with anti-Nuk antibodies which label the nerve fibers of the parasympathetic chain ganglion. Note the ganglia form a chain of interganglionic axonal connections with each other and that each ganglion unit forms connections with two segments of the neural tube.
  • Nuk protein localization within cranial nerve VII as it enters the second branchial arch can also be seen in Figure 8B.
  • Figure 8C shows a slightly different focal plane from (B) showing that the vestibulocochlear sensory fibers connecting to the developing ear stain positive for Nuk protein (arrow).
  • Nuk protein assodated with the developing eye is also shown in Figure 8C.
  • Transverse section of an 11.5 day embryo detects high levels of Nuk protein localized to the basement membrane of the endolymphatic duct cells where they contact the surrounding mesenchymal /neural crest cells (open arrow, Figure 8D).
  • Nuk protein associated with the vestibulocochlear ganglion is also visible in this section (filled arrow).
  • the scale bars in Figure 8 represent the following: (8A), 100 ⁇ m; (8B and 8C), 200 ⁇ m; (8D), 100 ⁇ m.
  • Nwfc 1 A loss of function mutation in Nuk, designated Nwfc 1 was generated in embryonic stem cells, and germline transmission of the null allele was obtained as described in more detail below.
  • null allele was generated in mouse embryonic stem cells generallly following the methodology described in Capecchi M.R., Science 244:1288-1292, 1989.
  • the null mutation was obtained by deletion of exon 2, corresponding to codons 29 to 50, as shown in Figure 9.
  • Nuk+I- embryonic stem cell lines ES
  • 8 cell embryos in vitro and the resulting blastocysts were transferred into redpient females.
  • animals chimeric for ES and embryonic stem cells were recovered by scoring for eye pigment and coat colour. Breeding of these "aggregation chimeras" confirmed that the germ line of at least one founder mouse is derived completely from the ES cells.
  • Adult mice homozygous for the mutation did not express Nuk protein.
  • EXAMPLE 9 Generation of a Nuk-lac Z fusion chimeric receptor mutant
  • a targeted mutation, designated Nuk 2 was generated in the Nuk gene as shown in Figure 10.
  • a pPNT-LOX-Nuk 2 gene trap vector was used to delete the GXGXXG ATP binding region of the kinase domain (amino acids 623-707, SEQ ID NO:2 and Figure 2) to create a Nuk-lac Z fusion receptor in ES cells.
  • Chimeric animals were prepared as described above, by aggregating the ES cells with 8 cell CD1 embryos. Animals generated with the Nuk 2 mutation provided Nu k expressing cells staining for ⁇ -galactosidase activity, providing a convenient marker for Nuk-positive cells in both heterozygous and homozygous backgrounds.
  • the Nu k 2 mutation led to the expression of a Nuk-lac Z fusion protein in mouse heterozygous embryos, detected by a blue/green colour as shown in Figures 11 and 12.
  • Figure 11 A shows an embryo at the six somite stage (8.25 days development) expressing the Nuk 2 mutation in the brain and developing heart.
  • Figure 11B shows that at the 14 somite stage (8.75 days development) expression continues in the hindbrain rhombomeres, the midbrain and diencephalon and persists in the heart.
  • Figure 12 shows Nu k 2 expression in a 10.5 day old embryo.
  • Figure 12A illustrates the very high levels of expression in the ventral midbrain, dienchephalon and retinal cells (which are out of focus in the photomicrograph).
  • Figure 12B illustrates expression in the brain and spinal cord.
  • EXAMPLE 10 Autophosphorylation of Nuk protein by ELK-ligand Fusion proteins consisting of the extracellular domain of Elk-ligand (Davis, S., et al. Sdence Vol. 266, Nov. 4, 1994, p.816) linked to the Fc portion of human immunoglobulin Gl were made in COS cells S/N following the methods outlined in Davis et al, 1994, supra. The expressed ligand was aggregated with anti-human Fc antibody and incubated with COS cells for 5 minutes, 15 minutes, 30 minutes or 1 hour. Following incubation, the COS cells were lysed and the lysate was immunoprecipitated with anti-Nuk antibodies.
  • Nuk expression in Cell Lines Various cell lines were screened for expression of Nuk protein. Cells were homogenized and the homogenate was incubated with anti-Nuk antibody and the immunopredpitates were collected by centrifugation. The washed immunopre ⁇ pitates were separated by SDS-polyacrylamide gel electrophoresis and subjected to Western blotting with anti-Nuk antibodies or anti- phosphotyrosine antibodies. As shown in Figures 14 and in Table 1, COS cells expressed high levels of Nuk protein.
  • Example 12 The experiments described in Example 12 were also carried out in the pre se n ce of a com petin g ph o sph o r yl ated pe pti de - GMKTpYIDPFTpYEDPNEAVR(K) from the Nuk membrane proximal region.
  • Nuk from COS cells obtained with Nuk antisera was phosphorylated by incubation with ATP.
  • the phosphorylated Nuk was incubated with bacterial lysates from SH2-GST fusion protein expressing bacteria, in the presence or absence of the competing peptide.
  • Western blots for were prepared using anti-GST antibodies.
  • ORGANISM Mus musculus
  • CTCTACTACT ATGAGGCTGA TTTTGACTTA GCCACCAAAA CCTTTCCCAA CTGGATGGAG 420
  • CAAGCAGCAC CATCGGCCGT GTCCATCATG CACCAGGTGA GCCGCACTGT GGACAGCATC 1380
  • GTCACTGTGC AGGGCCTCAA AGCCGGCGCC ATCTATGTCT TCCAGGTGCG GGCACGCACC 1560
  • GTCTTCCTCA TCGCTGTGGT CGTCATTGCC ATCGTATGTA ACAGACGGGG GTTTGAGCGT 1740
  • GGCATGAAGT ACCTGGCGGA CATGAACTAC GTGCACCGTG ACCTTGCTGC TCGAAACATC 2280 CTCGTCAACA GTAACCTGGT GTGTAAGGTG TCTGACTTTG GGCTCTCACG CTTCCTGGAG 2340
  • ORGANISM Mus musculus
  • Lys Val Asp Thr lie Ala Ala Asp Glu Ser Phe Ser Gin Val Asp Leu 145 150 155 160

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Abstract

Nouvelle protéine de tyrosine-kinase réceptrice et isoformes de celle-ci exprimées dans les axones en migration, et molécules d'acide nucléique codant les nouvelles isoformes de cette protéine et des parties de celles-ci. On a également prévu des procédés d'identification de substances aptes à se lier à la protéine réceptrice et des procédés de dépistage d'agonistes et d'antagonistes de la liaison entre la protéine et la substance. En outre, on a prévu des procédés diagnostiques et thérapeutiques mettant en ÷uvre cette protéine et les molécules d'acide nucléique.
EP95917841A 1994-04-29 1995-04-28 Tyrosine-kinase receptrice neuronale Withdrawn EP0804589A1 (fr)

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PCT/CA1995/000254 WO1995030326A1 (fr) 1994-04-29 1995-04-28 Tyrosine-kinase receptrice neuronale

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US6992175B1 (en) 1994-04-15 2006-01-31 Amgen Inc. Nucleic acids encoding Eph-like receptor tyrosine kinases
JPH11515105A (ja) * 1995-10-13 1999-12-21 マウント・サイナイ・ホスピタル・コーポレイション 新規リガンド調節経路の活性化方法
WO1998001548A1 (fr) * 1996-07-05 1998-01-15 Mount Sinai Hospital Corporation Recepteurs oligomerises modulant des voies regulees par des ligands transmembranaires pour des recepteurs tyrosine-kinases type elk
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